The problem of partially or completely removing the particles from a dispersion which contains particles dispersed in a carrier medium occurs in various fields of application. An especially important field is analytical methods for determining the concentration of components in blood. Such blood tests may in many cases not be performed with whole blood which contains the corpuscular components (blood corpuscles). Rather, it is necessary to obtain, from the whole blood, plasma which is as free as possible from cellular material.
The invention is, however, also suitable for treating other dispersions. The carrier medium must not be liquid, but rather can also be gaseous. An example of the application of the invention in the framework of diagnostic-analytic methods, in which a non-biological liquid is treated, is the manipulation, enrichment, or isolation of microbeads which, because of their large renewable surface, have recently been increasingly used in combinational chemistry and molecular biology, for example. In addition, the invention may also be used in other fields of chemical process engineering and the food industry in order to separate particles from process streams. Further usage possibilities exist in biotechnological methods (removal and isolation of cell cultures from corresponding dispersions) and in the field of wastewater purification. Without restriction of the generality, reference will be made hereafter to the treatment of dispersions in liquids, mainly the separation of plasma from whole blood.
Traditionally, centrifugation methods have been used in order to obtain for blood tests plasma by separation of the cellular components. However, this is not suitable for modern miniaturized tests. This is particularly true for point-of-care testing in which an analysis element (in the form of a test strip, for example) that is as small and compact as possible contains all of the reagents and other agents necessary for performing the test, so that the sample liquid must only be brought into contact with the analysis element in order to determine the desired analytical result visually or with the aid of an analysis instrument on the basis of a physical change detectable on the analysis element (particularly a color change or a change of an electrical measurement variable).
In order to obtain plasma for tests of this type from relatively small blood volumes, filtration methods have been discussed and used with some success for many years. Different filter media, particularly microporous membranes and fiberglass matting, are used. Early examples of these filtration technologies are described in U.S. Pat. Nos. 3,791,933 and 4,477,575. A more recent example having a complex combination of membrane and fiberglass filters is the subject of U.S. Pat. No. 6,045,699.
In U.S. Pat. No. 5,922,210 a microcomponent is described which is to be used for the purpose of obtaining by microfiltration extremely small quantities of plasma in the range up to approximately 1 μl. In a silicon substrate microchannels are generated by etching. The separation of the blood corpuscles is achieved by a barrier channel having a depth of less than 0.1 μm, so that the blood corpuscles cannot flow through the barrier channel. The required feed channels and the barrier channel are produced in two sequential production steps. The extremely low depth of the barrier channel of less than 0.1 μm is determined by the duration of the etching procedure in an etching bath. In consideration of the required high reproducibility this production method is very difficult and complex.
The above-mentioned methods for obtaining plasma have significant disadvantages. Above all, there is a high risk that the fine pores are clogged due to mechanical wear or the adhesion of cellular material to the pore walls. The filter capacity is thus limited.
A larger capacity requires a larger space for the filter media. In addition, the relation between the sample volume applied and the plasma volume obtained is unfavorable. Finally, measurement errors may be caused by adhesion of proteins to the filter medium or by the high shear forces during the passage of erythrocytes through the narrow filter pores and by hemolysis resulting therefrom.
On this basis, it is an object of the invention to allow separation of particles from a dispersion, while avoiding, as far as possible, the disadvantages described above, using a separating module which may be produced easily and cost-effectively. The separating module is preferably a “disposable”, intended for single use, and should in particular be suitable for generating small amounts of plasma (less than 10 μl, in particular less than 5 μl) for miniaturized tests.
The object is achieved by a method for separating particles from a fluid dispersion, particularly for separating corpuscular components from biological samples, above all from blood, by means of a separating module comprising a substrate with flow channels, including a feed channel for supplying the dispersion to a junction, a first drain channel for draining fluid having a reduced particle concentration away from the junction, and a second drain channel for draining fluid having an increased particle concentration away from the junction, wherein the fluid flows so much faster in the second drain channel than in the first drain channel that due to the different flow speeds the dispersed particles at the junction preferentially flow further in the second drain channel.
Earlier filtration methods used for the purposes of the invention are based on steric selection, i.e. on the fact that the particles to be separated are held back because the pores of the filter medium are smaller than the diameter of the particles. In order to separate erythrocytes reliably in this way, the pore diameter of the filter medium must be at most 1 μm (particularly because of the easy deformability of the erythrocytes).
In the invention, the selection is based on a completely different principle: differing local particle flow speeds in different flow paths of the liquid flow in the separating module lead to shear stresses which cause the particles to preferentially flow at the junction further in the second drain channel having the higher flow speed. The first flow channel having the lower flow speed contains a reduced particle concentration.
A plurality of important advantages are achieved by the invention:                Since the separation of the particles is not based on steric selection, the smallest dimension of the drain channels may be larger than the particle diameter. For example, the flow channels of a separating module suitable for obtaining plasma from whole blood preferably have a smallest cross-sectional dimension of at least 5 μm and at most 150 μm. Values of less than 100 μm, particularly less than 50 μm, are especially preferred. In this way, there is, in contrast to the previously known filtration methods, practically no risk of clogging of the filter medium. An additional advantage is due to the fact that no fibrous materials must be used, which cause additional clogging risk.        According to the invention, blood (or other dispersions) may be treated continuously over long periods of time. The separating module may therefore be used for continuously obtaining (practically) particle-free filtrates or for continuous particle enrichment from dispersions.        The manufacturing is relatively simple and inexpensive. In comparison to previously known filtration methods, it is not necessary to manufacture and integrate a filter medium into the separating module. In comparison to the microfilter described in U.S. Pat. No. 5,922,210, the manufacturing is significantly simpler because the flow channels integrated into the chip have comparatively large dimensions. Such channel structures may be produced cost-effectively in mass production. An especially suitable method includes production of a master by a photolithographic way. A mold may be obtained from this master, and from this mold product chips may be produced by pressing or injection molding (example: production of CDs). Smaller production lots may be produced by laser ablation.        It is advantageous for the production that the invention does not require structures of differing depths. Preferably, at least both drain channels, especially preferably all flow channels, are equally deep. They may be produced easily in a single work step.        The dead volume in the flow channels of the separating module is very small. The invention therefore allows a sufficiently large volume of plasma to be obtained from a very small sample volume.        The separating module according to the invention can be further miniaturized than a system which contains a filter medium and drain channels, and miniaturization does not reduce the efficiency of the separation or the throughput. This again helps to reduce the cost.        The separating module may be integrated easily into a system, particularly an analysis system. In analytical Microsystems, for example, “planar integration” is possible, i.e. reagents and liquid treatment elements necessary for the analysis can be integrated into the same chip in which the flow channels of the separating module are located. However, conventional coupling to an analysis system via tubing lines having a low dead volume is also possible.        
The physical effects upon which the invention is based may be partially explained on the basis of experimental investigations of the flow behavior of blood in the capillary system of the body and theoretical considerations based thereon. The available knowledge is summarized, for example, in a review article by A. R. Pries et al., “Biophysical aspects of blood flow in the microvasculature”, Cardiovascular Research 32, 1996, 654-667. The authors report inter alia that, at junctions of the capillary vessels transporting the blood in the body, the hematocrit (content of red blood corpuscles) is typically lower in a daughter vessel having a lower blood flow than in a daughter vessel having a higher blood flow. The statement is made that this phase separation can only insufficiently be described theoretically because of the numerous influencing variables and the dependence of the blood flow on these influencing variables, which is non-linear in multiple aspects. Specifically, the “plasma skimming effect”, the “network Fahraeus effect”, and the “pathway effect” are discussed as physical principles which determine the phase separation in capillary blood vessels. One of these effects, the network Fahraeus effect, describes the tendency of red blood corpuscles to preferentially follow at a junction the flow path having the higher flow rate (and therefore the higher flow speed).
According to the state of knowledge of the inventors, it is to be assumed that this principle essentially explains the function of the separating module according to the invention. It could not be expected, however, that a nearly complete plasma separation could be achieved in a practically usable manner by easily implementable means. This statement is confirmed by the fact that the basic principles about phase separation at capillary junctions have been known since a long time. For example, experimental in vitro investigations of 1964 and in vivo studies of 1970 are cited in the cited review article.
Another reason why the suitability of this principle for plasma separation was not to be expected is that in the natural capillaries no high degree or even complete separation is observed. In contrast, the functioning of the human body is dependent on a sufficiently high concentration of erythrocytes in even the finest capillaries to provide a sufficient oxygen supply. A further fundamental difference is that in the living body blood flows through a network of vessels having elastic walls with flow speeds which vary strongly in the rhythm of the blood pulse, while the liquid in a separating module flows at constant speed between rigid walls.
Evidently, it is not possible to derive from publications about the flow behavior in blood capillaries an indication that and how a practically useful separating module can be produced. Of special significance for the practical success of the invention is a preferred embodiment according to which the depth of the feed channel, preferably also the depth of the first drain channel and especially preferable the depth of all flow channels, is larger than the width, at least in the channel section directly adjoining the junction. This preferred embodiment relates to the fact that the separation of the particles is essentially determined by the width of the channels in the immediate neighborhood of the junction. By a depth which is large in relationship to the width, the separating performance (liquid volume separated per unit time) may be improved without impairing the function.
The invention will be described in greater detail here-after on the basis of exemplary embodiments shown in the figures. The features and elements shown and described may be used individually or in combination to provide preferred embodiments of the invention. In the figures: